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EP0871854B1 - Medium zum nachweis von enterococci in einer probe - Google Patents

Medium zum nachweis von enterococci in einer probe Download PDF

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Publication number
EP0871854B1
EP0871854B1 EP95938833A EP95938833A EP0871854B1 EP 0871854 B1 EP0871854 B1 EP 0871854B1 EP 95938833 A EP95938833 A EP 95938833A EP 95938833 A EP95938833 A EP 95938833A EP 0871854 B1 EP0871854 B1 EP 0871854B1
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EP
European Patent Office
Prior art keywords
medium
sample
enterococci
nutrient
detectable
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EP95938833A
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English (en)
French (fr)
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EP0871854A2 (de
Inventor
Chun-Ming Chen
Haoyi Gu
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Idexx Laboratories Inc
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Idexx Laboratories Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/10—Enterobacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/885—Streptococcus

Definitions

  • This invention is in the field of chemistry, biology and microbiology and relates to novel means for detecting the presence of target microbes in a sample of a possibly contaminated material.
  • Microorganisms are present ubiquitously in biological specimens and environmental media suitable for their growth. However, some prove harmful to higher organisms and means for detecting their presence is important to preserve the public health. Many means for detecting various types of microorganisms are available offering various advantages with respect to speed and specificity.
  • microorganisms have certain requirements for growth and reproduction.
  • microorganisms require the presence of the following for growth: an energy source including light and carbon compounds; and a source of raw materials including carbon, nitrogen, sulfur, and phosphorous as well as trace amounts of other minerals.
  • an energy source including light and carbon compounds
  • a source of raw materials including carbon, nitrogen, sulfur, and phosphorous as well as trace amounts of other minerals.
  • microorganisms must be present in a suitable environment wherein an appropriate temperature, pH, salinity and oxygen concentration is maintained.
  • a common procedure used to detect the presence of microorganisms involves adding a specimen to a culture medium containing all the necessary elements to allow growth.
  • the sample may be natural or pretreated, as by membrane filtration, before being added to the culture medium, and the medium may or may not contain chemicals such as antimetabolites or antibiotics which are selectively active against microorganisms other than the target microorganism.
  • these culture media have been sterile to assure no interference from contaminating microbes, and usually a rather long incubation step of from 48 to 72 hours has been required to provide for appropriate detection of Enterococci.
  • the target microorganism must be confirmed using one or more of a number of tests specific for a variety of physical and biochemical characteristics. These procedures are therefore labor intensive and time consuming.
  • Patent No. 3,496,066 describes the use of compounds which bacteria convert to dyestuffs.
  • B-ochner, U.S. Patent No. 4,129,483 describes using a non-biodegradable substance which is reduced to produce a color change.
  • the indicator is an additional substance and not one which also serves as a source of a required nutrient.
  • Enterococcus density is a predictor of public health risks associated with contaminated recreation waters.
  • MPN most probable number technique
  • MF membrane filter technique
  • the results based on the multiple-tube technique may not be available for 72 hours, and the results of the membrane filter technique may not be available for 48 hours.
  • the "MPN procedure” involves a 24 to 48 hour presumptive test in a series of azide dextrose broth followed by a 48 hour confirmation test using selective Enterococcus agar and 6.5 5 % NaCl brain-heart infusion broth.
  • the membrane filter technique involves the membrane filtration of water samples followed by incubation of a pre-filtered sterile membrane on Enterococcus selective media.
  • the media of choice are either mE agar followed by an EIA substrate test, or mEnterococcus agar. Such methods may be tedious, labor intensive and time consuming. This may lead to delays in public notification and therefore increase public health risks.
  • Panosian & Edberg Journal of Clinical Microbiology 27: 1719-1722 (1989) describes a method for detection of S. Bovis and related bacteria that comprises growing colonies of organisms on plates for periods of 24 to 48 hrs and 18 to 24 hrs followed by transfer of cultured colonies to a detection media comprising p-nitrophenyl- ⁇ -D-galactopyranoside and 4-methylumbilliferyl- ⁇ -D-glucoside.
  • Trepeta & Edberg, Antonie van Leeuwenhoek 53: 273-277 (1987) describe a method for detecting bacteria possessing esculinase and utilises a detection media comprising p-nitrophenyl- ⁇ -D-glucopyranoside.
  • the present invention features a medium which allows detection of Enterococci microbes in a liquified environmental or biological sample, within as little as 24 hours.
  • the present medium is distinct from prior media in which about 48 to 72 hours are required to obtain a test result for Enterococci.
  • the medium also allows quantifying the amount of Enterococci present in a sample and may be used in rapid screening methods.
  • the medium contains an effective amount of vitamin, amino acid, trace element and salt ingredients operable to allow viability and reproduction of Enterococci in the presence of a nutrient indicator.
  • the nutrient indicator is provided in an amount sufficient to allow a detectable characteristic signal to be produced in the medium by the growth of Enterococci.
  • the medium further contains effective amounts of selective agents which are active to prevent or inhibit the growth of non-target (i.e., non-enterococcal) microorganisms.
  • Media which have proven optimal in this invention for the detection of Enterococci in a sample include (per liter) a biological buffer (e.g., about 5.0 to 7.0 grams N-tris[Hydroxy-methyl]methyl-3-amino-propanesulfonic acid, free acid (TAPS-free acid), and about 5.0 to 7.0 grams N-tris [Hydroxy-methyl]methyl-3-amino-propanesulfonic acid, sodium salt (TAPS-sodium) or about 4.0 to 5.0 grams HEPES free acid and about 7.0 to 9.0 grams HEPES sodium salt)); and sodium bicarbonate (e.g., about 1.5 to 2.5 grams).
  • a biological buffer e.g., about 5.0 to 7.0 grams N-tris[Hydroxy-methyl]methyl-3-amino-propanesulfonic acid, free acid (TAPS-free acid), and about 5.0 to 7.0 grams N-tris [Hydroxy-methyl]methyl-3-amino
  • the following components are provided in the media in approximately the amounts indicated. Those in the art will understand that not every component is required. Components may also be substituted with other components of similar properties. The amounts of components may also be varied. Specifically, the medium contains (per liter) a total nitrogen content of about 1.0 to 1.7 grams (primarily from ammonium sulfate). Amino acids required for growth of the microorganisms to be detected are also provided. Not all amino acids must be provided, and the relative amount of each can vary. Amino acids may be provided from a variety of sources. These can be provided from natural sources ( e.g. , extracts of whole organisms), as mixtures or in purified form. The natural mixtures may contain varying amounts of such amino acids and vitamins.
  • amino acids and vitamins are indicated below. Those in the art will recognize that many different combinations of amino acids and vitamins can be used in media of this invention. Normally, only amino acids which cannot be synthesized endogenously by the microorganisms to be detected must be provided. However, other amino acids may be provided without departing from the medium of the invention.
  • Amino acid contents are at least the following in approximately the following amounts (per liter): Alanine (0.015 to 0.055 grams), Arginine (0.01 to 0.04 grams), Aspartic Acid (0.04 to 0.10 grams), Cystine (0.001 to 0.005 grams), Glutamic Acid (0.05 to 0.15 grams), Glycine (0.01 to 0.03 grams), Histidine (0.010 to 0.06 grams), Isoleucine (0.01 to 0.10 grams), Leucine (0.03 to 0.08 grams), Lysine (0.03 to 0.07 grams), Methionine (0.01 to 0.04 grams), Phenylalanine (0.01 to 0.04 grams), Proline (0.02 to 0.08 grams), Serine (0.01 to 0.05 grams), Threonine (0.01 to 0.04 grams), Tryptophan (0.002 to 0.006 grams), Tyrosine (0.01 to 0.02 grams), and Valine (0.02 to 0.05 grams).
  • Salts may be provided as a source of ions upon dissociation.
  • Such salts may include potassium phosphate ( e.g., about 0.5 to 1.5 grams); copper sulfate ( e.g., about 40 to 50 ⁇ g); ammonium sulfate ( e.g., about 4.0 to 6.0 grams); potassium iodide ( e.g. , about 50.0 to 150.0 ⁇ g); ferric chloride ( e.g., about 150.0 to 250.0 ⁇ g); manganese sulfate ( e.g., about 300.0 to 500.0 ⁇ g); sodium molybdate ( e.g. , about 150.0 to 250.0 ⁇ g); zinc sulfate ( e.g.
  • inorganic moieties may be included to aid in microbe growth. These include the following (to the extent not already provided in the above sources of various chemical entities and described in amounts per liter): Phosphorus (about 0.0005 g/l), Potassium (about 0.0004 g/l). Sodium (about 0.03 to 0.06 g/l), and trace amounts of Calcium, Magnesium, Aluminum, Barium, Chloride, Cobalt, Copper, Iron, Lead, Manganese, Suffate, Sulfur, Tin and Zinc.
  • Vitamins required for growth and reproduction of the microorganism sought to be detected may also be provided. These can be provided in a pure form or as part of more complex media. Such vitamins may be present in approximately the following amounts (per gram of medium): Biotin (about 0.15 to 0.40 ⁇ g/l) , Pantothenic Acid (about 45.0 to 65.0 ⁇ g/l), Pyridoxine (about 6.0 to 9.0 ⁇ g/l), Riboflavin (about 10.0 to 20.0 ⁇ g/l), Folic acid (about 5.00 to 10.00 ⁇ g/l), Thiamine (about 10.0 to 20.0 ⁇ g/l), Vitamin B12 (about 0.20 to 0.30 ⁇ g/l), and Niacin (about 15.0 to 25.0 ⁇ g/l).
  • Biotin about 0.15 to 0.40 ⁇ g/l
  • Pantothenic Acid about 45.0 to 65.0 ⁇ g/l
  • Pyridoxine about 6.0 to 9.0 ⁇ g/l
  • Riboflavin about 1
  • Selective agents and in particular antibiotics, which inhibit or prevent growth of non-target organisms may also be provided.
  • Many selective agents may be provided, and the selective agents used depends upon the target microorganism.
  • the selective agents include one or more of the following in concentrations within the following ranges: amikacin sulfate (about 0.0045 to 0.0055 grams/liter), amphotericin B (about 0.00198 to 0.00242 grams/liter), and Bacitracin (about 0.000476 to 0.00794 grams/liter).
  • amikacin sulfate about 0.0045 to 0.0055 grams/liter
  • amphotericin B about 0.00198 to 0.00242 grams/liter
  • Bacitracin about 0.000476 to 0.00794 grams/liter
  • thallium acetate, neomycin sulfate, cycloheximide, tetracycline, colistin, ansiomycin or clindamycin may be substituted.
  • the nutrient indicator is present in the medium in an amount which is sufficient to support growth of the target microbe until a detectable characteristic signal is produced in the medium during growth.
  • the vitamin, amino acid, trace element, salt and nutrient indicator ingredients allow sufficient growth of the organism so that a detectable change in the sample may be observed.
  • the nutrient indicator alters a detectable characteristic of the sample only when it is metabolized by an organism. Preferably, it alters a detectable characteristic only when it is metabolized by the target microbe. Therefore, it may be used to confirm the presence or absence of the target microbe in the sample. It is preferable that the nutrient indicator be chosen so that it is cleaved to release the indicator portion only by the target microbe in the medium.
  • the medium is formed such that such other microbes cannot substantially grow in the medium.
  • the nutrient indicator is the only source of the specific type of nutrient for the target microbe
  • the medium may contain other such sources, but in amounts that will not reduce the specificity of the medium.
  • the nutrient indicator may be the only source of carbon for this microorganism.
  • other carbon sources may be present (e.g. amino acids) which are not preferentially used by the target microbe.
  • the nutrient indicator is 4-methylumbelliferyl- ⁇ -D-glucopyranoside.
  • nutrient indicators which may be used in the invention include the following: 5-bromo-4-chloro-3-indoxyl- ⁇ -D-glucopyranoside, o-nitrophenyl- ⁇ -D-glucopyranoside, p-nitrophenyl- ⁇ -D-glucopyranoside, resofuran- ⁇ -D-glucopyranoside, 6-bromo-2-naphthyl-D-glucopyranoside, Rose- ⁇ -D-glucopyranoside, VQM-GIc (2- ⁇ 2- [4- (D-glucopyranosyloxy) -3-methoxyphenyl]vinyl) -1-methyl-guinolinium iodide, and VBzTM-Gluc(2- ⁇ 2-[4-(-D-glucopyranosyloxy)3-methoxyphenyl]vinyl ⁇ -3-methylbenzothiazolium iodide.
  • Enterococci includes the following microorganisms: Enterococcus avium, E. casseliflavus, E. cecorum, E. columbae, E. dispar, E. durans, E. faecalis, E. faecium, E. gallinarum, E. hirae, E. malodoratus, E. mundtii, E. psudoavium, E. raffinosus, E. saccharolyticus, E. seriolicida, E. solitarius, and E. sulfureus. Among them, E. avium, E. durans, E. faecalis, E. faecium, E.
  • gallinarum, and E. hirae are the strains of fecal origin. The term is not limited to mean any given number of these species and is not meant to exclude species which have yet to be discovered but may later be identified and included in this genus by those of skill in the art.
  • fecal streptococci includes species of the Streptococci bacteria present in the gastrointestinal tract of higher organisms. It includes such organisms as S. faecalis, S. faecuim, S. avium, and S. gallinarum. S. faecalis, S. faecium, S. avium, and S. gallinarum are more commonly referred to as Enterococci and are included within that term herein.
  • the term "24 hours” means the time required for about 95% of the liquid samples containing only about one to ten Enterococci per 100 ml to display a detectable characteristic change.
  • the temperature, amount and type of enzyme inducer present, amount of nutrients provided in the medium, and the relative health of the bacteria all affect the detection time.
  • the amount of nutrients such as amino acids, vitamins and trace elements provided may affect growth rate of the target microbe and thus detection time.
  • Thermally equilibrating the sample to an incubation temperature of about 35°C after adding the medium may decrease the time required for detection.
  • the amount of enzyme inducer may also decrease the time to detection.
  • Enzyme inducers found in the medium are agents which act as an inducer of the enzyme which cleaves the nutrient indicator.
  • the enzyme inducer may, for example, be a homolog to the nutrient indicator. Examples of such inducers are known in the art.
  • the relative health of the microbe also affects the time required for detection. Adding such agents as pyruvate which may aid recovery of injured organisms may therefore speed detection. If large numbers of bacteria are present in the sample, more rapid detection is also possible.
  • the media provided allows detection of low amounts of target microbes ( i.e. less than 10/100 ml) in the 24 hour time period, at least 95% of the time. Standard methods can be used to determine such ability.
  • medium means a solid, powder or liquid mixture which contains all or substantially all of the nutrients necessary to allow a microbe to grow and reproduce.
  • This invention includes both media which are sterilized as well as media which are not sterile.
  • liquid means substantially in liquid form, though it is also meant to include pulverized or homogenized samples of solid substances having at least a 10% liquid content.
  • the phrase is meant to exclude a gelled medium, such as is found with agar.
  • environment and “biological” mean taken from or coming from a substance capable of supporting one or more life forms including algae, yeast and bacteria. Examples include but are not limited to recreational waters, marine waters, drinking waters, sewage effluents, and food substances.
  • the term “inoculation” means at or near the time the liquified environmental or biological sample is mixed with the medium of this invention. It is meant to be the time at which the two substances are substantially mixed together.
  • the term "effective amount” is an amount within the range which allows or promotes growth and reproduction of a target microorganism. That is, an amount which allows microbes or other organisms to adapt to the medium, synthesize the necessary constituents for reproduction and subsequently reproduce. It is not meant to be specific and may vary depending upon such factors as sample size and concentration of microorganisms. Generally, the term indicates the amount required to detect less than 100 target microbes per 1 ml sample, most preferable less than 100 microbes per 100 ml sample, or even 1 microbe per 100 ml sample.
  • vitamins vitamins, amino acids, trace elements and “salts” are meant to include all molecules, compounds and substances classified in each category by those of skill in the art whether organic or inorganic, and the categories are not intended to exclude any substance which may be necessary for or conducive to maintaining life.
  • nutrient indicator means a molecule or substance containing a moiety that is a source for an essential nutrient and a moiety which causes or produces an observable characteristic change in the medium or sample.
  • a nutrient indicator includes nutrient sources attached to or conjugated with chromogens or fluorogens. Nutrient sources may provide essential vitamin, mineral, element or amino acid ingredients or carbon. Normally, as a microorganism progresses from the phase in which nutrients are accumulated for reproduction (lag phase) and into the phase during which reproduction actually occurs at a relatively rapid rate (log phase), nutrition requirements increase. Consequently, increased amounts of the nutrient indicator are metabolized and a detectable and characteristic change is produced.
  • the nutrient indicator includes a nutrient moiety and a chromogen or a flurogen.
  • Chromogens include any moieties which produce a color change observable in the visible range.
  • Fluorogens include any moieties which fluoresce upon exposure to an excitation light source. Examples include, but are not limited to, orthonitrophenyl, phenolphthalein, and 4-methylumbelliferone moieties. While the nutrient indicator may provide the sole source of an essential nutrient, other sources of such nutrients may be provided, so long as adequate selectivity and sensitivity of the medium is maintained.
  • detecttable characteristic signal includes any change in a sample which may be detected by one or more of the human senses.
  • the term includes such examples as a color change in the visible or non-visible wavelength ranges, a change in state such as between solid, liquid and gas, an emission of gas, or a change in odor.
  • target microbe means the microorganism whose presence or absence is sought to be detected.
  • it includes species of enterococci and fecal streptococci which can live in the gastrointestinal tract of higher organisms.
  • the nutrient indicator alters the color of the microbe-specific medium.
  • the color change may be apparent in the visible wavelength range or it may be fluorescence which is apparent in a wavelength range visible after exposure to an excitation light source. This is accomplished by the cleavage of a chromogenic moiety or fluorescent moiety.
  • a chromogenic moiety is a moiety which changes the color of the sample in the visible range when it is in an unconjugated form, that is no longer conjugated to or bound to a nutrient moiety.
  • a fluorescent moiety is a moiety which changes the color of the sample in the non-visible range when it is in an unconjugated form, that is no longer conjugated to or bound to a nutrient moiety.
  • chromogenic moieties that may be conjugated to a nutrient moiety include, but are not limited to orthonitrophenyl moieties which produce a yellow color when released from the nutrient indicator, and phenolphthalein moieties which produce a red color when released from the nutrient indicator.
  • fluorescent moieties include, methylumbelliferyl moieties which become fluorescent at about 366 nm when released from the nutrient indicator, and bromo-chloro-indole moieties which become blue when released from the nutrient indicator.
  • the medium uses the fluorescent moiety, 4-methylumbelliferyl- ⁇ -D-glucopyranoside.
  • the medium also contains one or more selective agents such as antibiotics which prevent or inhibit microbes other than the target microbe from metabolizing the nutrient indicator. That is, preferably the medium contains agents which are specific for microbes other than Enterococci or fecal Streptococci and effectively prevent or inhibit growth of at least some of those microbes.
  • the term is meant to include such agents as sodium azide, thallium acetate, nalidixic acid, neomycin sulfate, gentamicin sulfate, bile salt, sodium chloride, lycloheximide, tetracycline, colistin, ansiomycin, clindamycin, and polymycin B.
  • amikacin sulfate e.g. about 0.0045 to 0.0055 g/liter
  • amphotericin B e.g. about 0.00198 to 0.00242 g/liter
  • bacitracin e.g. about 0.000476 to 0.000794 g/liter
  • microbe-specific medium means a medium which allows substantial growth of only the target microbe. This includes media which contain one or more antibiotics specific for inhibiting growth of microorganisms other than the target microbe, and it includes media which alternatively or additionally contain one or more nutrient indicators which are preferably not metabolized by microorganisms other than the target microbe to any substantial degree.
  • substantially as used in this context, means that the medium still allows specific ( i.e., at least 95% or even 98% accurate) and sensitive ( i.e., at least 95% or even 98% detection levels) detection of the target microbe, as measured by standard procedures.
  • the medium contains an agent which acts as an inducer of the enzyme which cleaves the nutrient indicator.
  • This agent may, for example, be a homolog to the nutrient indicator.
  • inducers include isopropyl- ⁇ -D-thiogalactoside (IPTG) which induces ⁇ -galactosidase activity and ethyl- ⁇ -D-thioglucoside which induces ⁇ -glucosidase activity.
  • the medium allows detection of Enterococci (including fecal Streptococci) within 24 hours.
  • the medium is also preferably in the form of a non-sterile, water soluble powder to allow easy addition to liquid samples.
  • the invention features a method for detecting the presence or absence of Enterococci and fecal Streptococci in a liquified sample by contacting the sample with the medium described above, incubating the sample and the medium mixture, and monitoring the sample and medium mixture to determine the presence or absence of the detectable characteristic signal.
  • the incubation may be performed at a variety of temperatures, but preferably it is carried out between 35°C and 45°C.
  • the detectable characteristic signal may be observed within 24 hours.
  • the invention features providing samples preferably from a water source including fresh water, marine water, drinking water supplies or waste water.
  • Another feature of the invention is a method for detecting the presence or absence of a target microbe in an environmental or biological liquid sample, preferably including the step of warming the sample to incubation temperature in a liquid incubator after adding the microbe-specific medium.
  • the incubation temperature is about 35 °C.
  • liquid incubator means a liquid warmed to a specified temperature or temperature range. This may include any form of water baths for instance. Such an incubator is advantageous over previously used air incubators, since the medium more quickly reaches an adequate incubation temperature.
  • the invention features a method for quantifying the amount or number of Enterococci present in a liquid sample by contacting a liquid sample with the medium described above, placing the liquid sample including the medium in containers, incubating the liquid sample and medium mixture, observing the quantity and quality of a detectable characteristic signal, and comparing the quantity and quality of a detectable characteristic signal with most probable values.
  • This quantifying process features comparing the quantity and quality of the characteristic which has been altered, preferably a color change, to most probable number values obtained from samples where the concentration and characteristic change have been correlated with samples for which Enterococci or bacteria concentration is known. See e.g., Compendium of Methods for the Microbiological Examination of Foods 3rd ed. , Edited by Vanderzant and Splittstoesser, 1992.
  • the most probable number technique allows estimation of bacterial concnetrations that are below detectable limits of other methods.
  • the invention uses the apparatus described by Naqui et al. in U.S. Patent No. 5,518,892.
  • the quantifying step involves providing a sample of an environmental or biological sample in a liquid form, placing or dispensing the sample into the sample holding bag described by Naqui, mixing the sample with a medium to allow and promote growth of viable bacteria, incubating the sample, detecting the quantity and quality of the color change, and comparing that quantity and quality with results obtained for a series of samples for which the concentration of bacteria was known. More preferably, the incubation step is carried out at 41°C for a period of 24 hours using the medium described above.
  • the invention provides the optimal medium for determining the presence of Enterococci (including fecal Streptococci) microorganisms. Enterococci can be detected much earlier in this medium than in those currently available. Therefore, the results of testing are more rapidly available. Rapid results save both money and time in the laboratory. Speed also decreases the threat to the public health, allowing early alerts and remedial measures to deal with the presence of some microorganisms in such places as drinking water supplies and recreational waters. Further, the method of this invention generally does not require confirmatory tests since microorganism-specific nutrient indicators may be used. Additionally, the invention does not require using a sterile medium as many other methods require.
  • compositions, methods, and products of this invention are applicable to biological and environmental specimens, and are useful in the chemical, biological and microbiological arts for the detection of microorganisms.
  • microorganisms derive their nutrients from an array of sources, however, some sources may be unique to a particular microorganism or group of microorganisms. Families, groups or species of microorganisms may share enzyme specificity for certain nutrients which is lacking in other microorganisms. By taking advantage of the metabolic characteristics of specific microorganisms, it is possible to test for their presence. Many enzymes have been identified which are specific to particular groups or species and others likely will be identified in the future.
  • the enterococcus group of bacteria contain a unique constitutive enzyme, ⁇ -glucosidase (Littel, et al., Appl. Environ. Microbiol. 45:622-627 (1983). It catalyzes the hydrolysis of appropriate chromogenic or fluorogenic substrates under appropriate selective environments resulting in a colored or fluorescent signal that can be detected either visually or spectrophotometrically.
  • ⁇ -glucosidase substrate may serve as the nutrient indicator in media designed to detect enterococci and provide a major source of carbon in the medium formulation. A number of nutrient indicator substrates are available.
  • the substrate preferably used in detecting enterococci is the fluorogenic substrate, 4-methylumbelliferyl- ⁇ -D-glucopyranoside.
  • the nutrient indicator is metabolized thereby cleaving the indicator portion which, when cleaved, becomes fluorescent.
  • the glucose moiety released is then utilized by enterococci bacteria to promote growth.
  • enterococcus and fecal streptococcus group of bacteria is a valuable bacterial indicator for determining the extent of fecal contamination in recreational surface waters (Greenberg, et al., Standard Methods for the Examination of Water and Wastewater. 18th ed. Eaton, A.D. (ed.) American Public Health Association (1992)).
  • the genus Enterococcus now contains 18 species: Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus, Enterococcus saccharolyticus, Enterococcus seriolicida, Enterococcus solitarius, and Enterococcus sulfureus.
  • Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, and Enterococcus hirae are the stains of fecal origin (Hernandez, et al., Wat. Res. 27:597-606 (1993)). These bacteria survive much longer than other indicators in marine environments. Enterococci are also resistant to sewage treatment, including chlorination, and thus are sensitive indicators of the survival of enteric pathogens and viruses in water samples. A direct correlation between the concentration of enterococci in marine waters and the risks of swimming-associated gastroenteritis has been demonstrated (Cabelli, Wat. Sci. Tech.
  • enterococci group of bacteria be used as the bacterial indicators for fresh and marine waters.
  • Current safety guidelines for recreational waters based on enterococci density are 33 enterococci per 100 ml for fresh water and 35 enterococci per 100 ml for marine water, respectively.
  • ⁇ -glucosidase activity is a specific characteristic for detecting enterococci and fecal streptococci.
  • Other bacteria also possess such enzyme activity. These include the genera of the family of Enterobacteriaceae ( Enterobacter aerogenes, E. colacae ) Klebsiella pneumoniae, Serratia marcescens, Listeria monocytogenes, and Bacterioides fragilis. Certain selective factors(s) may be used to inhibit the growth of such other ⁇ -glucosidase positive bacteria ( i.e. those other than enterococcus species).
  • a medium selective for specific microorganisms may be produced by utilizing a combination of enzyme specificity and selective environments. For instance, in one medium of this invention, non-target microbes which do not possess ⁇ -glucosidase activity and cannot digest the nutrient indicator are suppressed. Heterotrophic bacteria or other non-target microbes that possess ⁇ -glucosidase are selectively suppressed during the test period by the combination of specifically formulated chemical/antibiotic reagents and other physical parameters (pH and temperature).
  • Typical selective agents that can be used in the medium of this invention include sodium azide, thallium acetate, nalixidic acid, neomycin sulfate, gentamicin sulfate, bile salt, sodium chloride, and polymycin B (Hernandez et al., Wat. Res. 27:597-606 (1993); Knuntson, et al., Appl. Environ. Microbiol. 59:936-938 ((1993); and Littel et al., Appl. Environ. Microbiol. 45:622-627 (1983) ) .
  • the combination of enzyme specificity and antibiotic selectivity provides multiple hurdles which prevent the competing non-enterococcus microbes from being detected within the test period of 24 hours. This avoids the use of a single, highly toxic element which not only may inhibit the non-target microorganisms but also suppress the target microbes.
  • ⁇ -glucosidase catalyzes the conversion of the fluorescent substrate, 4-methylumbelliferyl- ⁇ -D-glucopyranoside (MUD), to 4-methylumbelliferone and glucose.
  • the flurophore, 4-methylumbelliferone emits blue fluorescence when excited at 365 nm (which may be viewed by a UV 365nm-lamp).
  • the released sugar moiety, glucose serves as a major carbon source to support growth of Enterocicci.
  • An increased nutrient indicator level may provide better microbial growth and stronger fluorescence, however, high nutrient indicator levels may also cause cell cytotoxicity and a greater level of background fluorescence.
  • 4-methylumbelliferyl- ⁇ -D-glucopyranoside (MUD) at a level of 25 mg/l does not inhibit the growth of enterococcus species.
  • glycolytic hydrolases ⁇ -glucosidase, ⁇ -galactosidase, and ⁇ -glucuronidase are inducible.
  • IPTG isopropyl- ⁇ -D-thiogalactoside
  • Ethyl- ⁇ -D-thioglucoside which functions in a similar fashion is a ( ⁇ -glucosidase inducer.
  • NaHCO 3 (2 g/liter) , Tween-80® (0.75 ml/liter), and KH 2 PO 4 (5 g/liter) stimulate growth of fecal streptococcus species isolated from waters (See, e.g. Lachica et al., J. App1. Bacteriol. 31:151-156 (1968)).
  • Other trace elements such as specific amino acid(s) (glutamic acid), vitamin(s) (lipoic acid), and nucleotide(s) (kinetin riboside) may all have growth promoting activities for enterococcus species.
  • the medium of the invention for detecting Enterococci is described in Table 1.
  • the medium of this invention can be used in three different formats. First, it may be used to detect the presence or absence of Enterococci. Second, it may be used to quantify the amount of Enterococci present in a sample. Third, it may be used in a screening format to relate the time for a positive test result with the concentration of Enterococci in a sample.
  • a positive reaction can occur anytime within 24 hours if there are viable bacteria present in the sample. That is, about 95% % of samples containing 10 cfu/100 ml will have exhibited a detectable characteristic change within 24 hours.
  • the time for detection ranging from about 12 to 24 hours, varies with the concentration and presence of different species or strains of enterococci.
  • the same medium can be used for quantifying target molecules.
  • the assay can be done with the regular 5 or 10 tubes MPN format or the 50 to 100 wells " Quanti-tray " MPN format (See, Naqui, U.S. Patent Application 08/201,110).
  • Quanti-tray MPN format
  • the medium described above can also be used for the application of pass/fail rapid screening.
  • This format involves the use of a direct linear relationship between the enterococcus density in a tested water sample with the detection time of positive results.
  • the procedure for rapid pass/fail screening test using the same formula is followed:
  • Enterococcus strains used were grown on TSAI blood agar platters. The following microorganisms were grown: E. faecalis ATCC 19433, ATCC 33012, ATCC 33186, ATCC 35550, ATCC 29212; E. faecium ATCC 19434, ATCC 35667; E. durans ATCC 6056, ATCC 11576, ATCC 19432; E. avium ATCC 14025, ATCC 35665; E. gallinarium ATCC 49573; Streptococcus bovis ATCC 9809, ATCC 35034; and S. equinus ATCC 9812.
  • Cell suspensions were prepared by taking the cells from plates using a sterile cotton swab (prewetted) and resuspended in pH 7.5 50 mM HEPES buffer to a turbidity equivalent to MacFarland Standard.
  • the following enzyme substrates were tested for sensitivity to ⁇ -glucosidase : o-nitrophenyl- ⁇ -D-glucopyranoside (ONPD) and 4-methylumbelliferone ⁇ -D-glucopyranoside (MUD).

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Claims (16)

  1. Medium um das Vorhandensein oder Fehlen von Entrococci in einer verflüssigten nichtgellierten Probe festzustellen:
    a) mit Alanin (0,015 - 0,55 g/l), Arginin (0,01 - 0,04 g/l), Aspartinsäure (0,04 - 0,10 g/l), Cystin (0,001 - 0,005 g/l), Glutaminsäure (0,05 - 0,15 g/l), Glyzin (0,01 - 0,03 g/l), Histidin (0,010 - 0,06 g/l), Isoleuzin (0,01 - 0,10 g/l), Leuzin (etwa 0,03 -0,08 g/l), Lysin (etwa 0,03 - 0,07 g/l), Methionin (etwa 0,01 - 0,04 g/l), Phenylalanin (etwa 0,1 - 0,04 g/l) , Prolin (etwa 0,02 - 0,08 g/l), Serin (0,01 - 0,05 g/l), Threonin (etwa 0,01 - 0,04 g/l) , Tryptophan (etwa 0,002 - 0,006 g/l), Tyrosin (etwa 0,01 -0,02 g/l) und Valin (etwa 0,02 - 0,05 g/l);
       Biotin (etwa 0,15 bis 0,40 µg/l), Pantothensäure (etwa 45.0 - 65.0 µg/l), Pyridoxin (etwa 6.0 - 9.0 µg/l), Riboflavin (etwa 10,0 - 20,0 µg/l), Folsäure (5.00 - 10,00 µg/l), Thiamin (10,0 - 20,0 µg/l), Vitamin B12 (etwa 0,20 - 0,30 µg/l) und Niazin (15,0 - 25,0 µg/l);
       Phosphat (0,0005 - 0,005 g/l), Kalium (etwa 0,0004 - 0,004 g/l), Natrium (etwa 0,03 bis 0,06 g/l) und Spurenmengen von Calcium, Magnesium, Aluminium, Barium, Chlorid, Kobalt, Kupfer, Eisen, Blei, Mangan, Sulfat, Schwefel, Zinn und Zink;
    b) mit einer wirksamen Menge eines oder mehrerer Nährmittelindikatoren, die in einer solchen Menge beigefügt sind, dass sie ausreichen um ein erkennbares charakteristisches Signal in dem Medium während des Wachstums von Enterococci zu erzeugen, wobei der Nährstoff Indikator eine Nährstoffmenge in Anwesenheit von β-Glukosidase abgibt und
    c) mit einer wirksamen Menge eines oder mehrerer selektiver Wirkstoffe, die in der Lage sind, das Wachstum von anderen Mikroorganismen als Enterococci zu verhindern oder zu inhibieren,
       wobei das Medium einen erkennbaren charakteristischen Wechsel innerhalb von 24 Stunden zeigt, wenn in der Probe, ein bis zehn Enterococci pro 100 ml enthalten sind.
  2. Medium nach Anspruch 1, das ferner enhält: einen Puffer, 4,0 bis 6,0 g/l Amoniumsulfat, eine Quelle für Kohlendioxid und Phosphorionen, eine wirksame Menge eines β-glukosidaseinducers, eine wirksame Menge von Antibiotika, um das Wachsen von Pilzen und anderen grampositiven und gramnegativen Bakterien als Enterococci zu inhibieren.
  3. Medium nach Anspruch 1 oder 2, bei dem der Nährmittelindikator bis zu einer Menge metabolisiert ist, die das als Nährstoffquelle dient, und einer Menge, die ein beobachtbares Characteristikum des Mediums verändert.
  4. Medium nach einem der Ansprüche 1 bis 3, bei dem der Nährmtelindikator die Farbe des Mediums in einem sichtbaren Wellenlängenbereich in Folge der Wirkung der β-glukosidase-Aktivität verändert.
  5. Medium nach einem der Ansprüche 1 bis 3, bei dem der Nährmittelindikator die Farbe des Mediums in einem nicht sichtbaren Wellenlängenbereich in Folge der Wirkung der β-glukosidase-Aktivität verändert.
  6. Medium nach Anspruch 5, bei dem der Nährmittelindikator 4-methylumbelliferyl-β-D-glycopyranosid ist.
  7. Medium nach Anspruch 1, das ferner enthält: einen Puffer, 4,000 bis 6,000 g modifizierte Stickstoffhefebase, 1,0 bis 2,5 g/l Natriumbikarbonat, 0,01 bis 1,0 g/l Kaliumphosphat, 0,009 bis 0,011 g/l Ethyl-β-D-Thioglucosid, 0,02 bis 0,03 g/l 4-methylumbelliferyl-β-D-glycolpyranosid, 0,0045 bis 0,0055 g/l Amikazinsulfat, 0,00198 bis 0,00242 g/l Amphoterizin B und 0,00476 bis 0,00794 g/l Bacitrazin.
  8. Verfahren zum Erkennen des Vorhandenseins oder Fehlens von Enterococci in einer verflüssigten nichtgelierten Probe, mit den Schritten:
    a) eine Flüssigkeitsprobe wird mit dem Medium nach einem der Ansprüche 1 - 7 zusammengebracht,
    b) die Flüssigprobe und das Medium werden inkubiert und
    c) die Flüssigkeitsprobe wird beobachtet, um das Vorliegen eines erkennbaren Wechsels bei einem physikalischen Charakteristikum festzustellen,
       wobei das Medium einen erkennbaren Wechsel des Charakteristikums innerhalb von 24 Stunden im Falle einer Probe zeigt, die zwischen 1 bis 10 Enterococci pro 100 ml enthält.
  9. Verfahren nach Anspruch 8, bei dem der Beobachtungsschritt maximal 24-Stunden andauert.
  10. Verfahren nach Anspruch 8 oder 9, bei dem die Flüssigkeitsprobe aus einer Frisch- oder Meerwasserquelle entnommen ist.
  11. Verfahren nach Anspruch 8 oder 9, bei dem die Flüssigkeitsprobe aus einer Abwasserquelle entnommen ist.
  12. Verfahren nach Anspruch 8 oder 9, bei dem die Flüssigkeitsprobe aus einer Trinkwasserquelle entnommen ist.
  13. Verfahren nach einem der Ansprüche 8 bis 12, bei dem es sich bei dem erkennbaren Wechsel des physikalischen Charakteristikums um einen Farbwechsel handelt.
  14. Verfahren nach einem der Ansprüche 8 bis 13, bei dem es sich bei dem erkennbaren Wechsel des physikalischen Charakteristikums um einen sichtbaren Farbwechsels in Anwesenheit einer anregenden Lichtquelle handelt.
  15. Verfahren nach einem der Ansprüche 14, bei dem der Inkubationsschritt bei 35°C bis 45°C durchgeführt wird.
  16. Verfahren zum Quantifizieren der Menge oder Anzahl von Enterococci, die in einer flüssigen nicht gelierten Probe enthalten sind, wobei das Verfahren die Schritte aufweist:
    a) die flüssige Probe wird mit dem Medium nach einem der Ansprüche 1 bis 7 zusammengebracht,
    b) die Flüssigkeitsprobe und das Medium werden in einem Behälter untergebracht;
    c) die Mischung aus Medium und Flüssigkeitsprobe werden inkubiert;
    d) die Quantität und die Qualität eines erkennbaren Wechsels eines physikalischen Charakteristikum werden beobachtet;
    c) die Qualität und die Quantität des erkennbaren Wechsels des physikalischen Charakteristikums werden mit höchstwahrscheinlichen Zahlenwerten verglichen, wobei das Medium einen erkennbaren Wechsel des Charakteristikums innerhalb von 24 Stunden bei der Probe zeigt, die 1 bis 10 Enterococci pro 100 ml enthält.
EP95938833A 1994-11-04 1995-10-23 Medium zum nachweis von enterococci in einer probe Expired - Lifetime EP0871854B1 (de)

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US08/335,149 US5620865A (en) 1994-11-04 1994-11-04 Medium for detecting Enterococci in a sample
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PCT/US1995/013579 WO1996015435A2 (en) 1994-11-04 1995-10-23 Medium for detecting enterococci in a sample

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